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Article

Escherichia coli-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles

1
Department of Biochemical Engineering, University College London, London WC1E 6BT, UK
2
Division of Medicine, UCL Institute for Liver and Digestive Health, Royal Free Campus, London NW3 2PF, UK
*
Author to whom correspondence should be addressed.
Academic Editor: Jorge H. Leitão
Vaccines 2021, 9(3), 193; https://doi.org/10.3390/vaccines9030193
Received: 16 January 2021 / Revised: 15 February 2021 / Accepted: 22 February 2021 / Published: 25 February 2021
(This article belongs to the Special Issue New Vaccine Technologies and Approaches 2.0)
Tandem-core hepatitis B core antigen (HBcAg) virus-like particles (VLPs), in which two HBcAg monomers are joined together by a peptide linker, can be used to display two different antigens on the VLP surface. We produced universal influenza vaccine candidates that use this scaffold in an Escherichia coli-based cell-free protein synthesis (CFPS) platform. We then used the CFPS system to rapidly test modifications to the arginine-rich region typically found in wild-type HBcAg, the peptide linkers around the influenza antigen inserts, and the plasmid vector backbone to improve titer and quality. Using a minimal plasmid vector backbone designed for CFPS improved titers by at least 1.4-fold over the original constructs. When the linker lengths for the influenza inserts were more consistent in length and a greater variety of codons for glycine and serine were utilized, titers were further increased to over 70 μg/mL (4.0-fold greater than the original construct) and the presence of lower molecular weight product-related impurities was significantly reduced, although improvements in particle assembly were not seen. Furthermore, any constructs with the C-terminal arginine-rich region removed resulted in asymmetric particles of poor quality. This demonstrates the potential for CFPS as a screening platform for VLPs. View Full-Text
Keywords: cell-free protein synthesis; virus-like particle; tandem-core; influenza vaccine cell-free protein synthesis; virus-like particle; tandem-core; influenza vaccine
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MDPI and ACS Style

Colant, N.; Melinek, B.; Frank, S.; Rosenberg, W.; Bracewell, D.G. Escherichia coli-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles. Vaccines 2021, 9, 193. https://doi.org/10.3390/vaccines9030193

AMA Style

Colant N, Melinek B, Frank S, Rosenberg W, Bracewell DG. Escherichia coli-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles. Vaccines. 2021; 9(3):193. https://doi.org/10.3390/vaccines9030193

Chicago/Turabian Style

Colant, Noelle; Melinek, Beatrice; Frank, Stefanie; Rosenberg, William; Bracewell, Daniel G. 2021. "Escherichia coli-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles" Vaccines 9, no. 3: 193. https://doi.org/10.3390/vaccines9030193

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